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Abstract

Platinum nanoparticles (NP-Pt) are noble metal nanoparticles with unique physiochemical
properties that have recently elicited much interest in medical research. However,
we still know little about their toxicity and influence on general health. We investigated
effects of NP-Pt on the growth and development of the chicken embryo model with emphasis
on brain tissue micro- and ultrastructure. The embryos were administered solutions
of NP-Pt injected in ovo at concentrations from 1 to 20 μg/ml. The results demonstrate that NP-Pt did not
affect the growth and development of the embryos; however, they induced apoptosis
and decreased the number of proliferating cells in the brain tissue. These preliminary
results indicate that properties of NP-Pt might be utilized in brain cancer therapy,
but potential toxic side effects must be elucidated in extensive follow-up research.

Keywords:

Background

Platinum (Pt) is a noble metal with unique physiological and chemical properties widely
used in chemistry, physics, biology, and medicine. Regarding the biological activities
of Pt, it is known that Pt compounds have the ability to arrest the cell cycle [1,2] and cause DNA strand breaks. The DNA damage is caused by Pt ions, which attach to
N7 sites of DNA guanine bases and, after hydrolysis of Pt-Cl bonds, form adducts with
the DNA double helix [2,3]. These properties of Pt are exploited in cancer therapy in the form of antineoplastic
drugs to treat different types of cancer such as head, neck, brain [4], testicular, bladder, ovarian, or uterine cervix carcinomas [5]. However, toxic side effects of Pt-based drugs are major drawbacks in cancer therapy
[6,7].

Nanotechnology has introduced possibilities for using alternate forms of elements
- nanoparticles. Nanoparticles have unique physiochemical features because of their
small size (<100 nm), large surface-to-mass ratio, exceptional quantum characteristics
[8], and consequently unique biological properties. Smaller nanoparticles can move across
cellular and also nuclear membranes and are able to penetrate cells and intracellular
structures, and target defined points within the body [9,10]. Platinum nanoparticles (NP-Pt) have recently elicited much interest because of their
physicochemical properties such as catalytic activity and high reactivity [11]. NP-Pt, as metal structures (Pt0), differ significantly from platinum salts and have quite different chemical properties
when administered to an organism. They are a very limited source of ions, and consequently,
the process of forming platinum salts is very slow and restricted. However, the solubility
and, consequently, the bioavailability of NP-Pt depend on their size [12]. Although it has been demonstrated that small doses of NP-Pt have negligible toxic
effects on chicken and zebra fish embryos [13], they might impinge the cell structures [12].

It has been demonstrated that hadron cancer therapy can be amplified by simultaneous
application of NP-Pt, resulting in the production of hydroxyl radicals causing lethal
DNA damage by double-strand breaks [14]. Furthermore, DNA damage could also be induced by the attack of OH groups linked
with NP-Pt on DNA phosphate groups [2]. NP-Pt can also cause cell cycle arrest and induction of apoptosis through the release
of Pt2+ ions from the nanoparticles as a result of H2O2 generation due to the low pH in endosomes [1]. It was also demonstrated that DNA double-strand breaks are caused by Pt2+ ions formed during the incubation of NP-Pt with cancer cells [15]. However, the consequences of introducing NP-Pt into an organism are still not well
documented, especially when even very small amounts of nanoparticles or released ions
may overcome the blood–brain barrier (BBB), enter the brain tissue, and affect the
BBB and brain function. It has also been reported that various types of nanoparticles,
in different sizes from 20 to 300 nm and produced from different materials, may cause
cell death by apoptosis in the brain tissue [16].

In the present study, we hypothesized that NP-Pt may affect the growth and development
of embryos and, furthermore, can cross the BBB and penetrate the brain tissue, affecting
brain morphology. Consequently, the objective of this preliminary work was to investigate
the effects of NP-Pt on embryo growth and development with an emphasis on brain morphology,
concerning their potential applicability in brain cancer therapy.

Methods

Nanoparticles

Hydrocolloids of NP-Pt were obtained from Nano-Tech Polska (Warsaw, Poland). They
were produced by a patented electric nonexplosive method [17] from high purity metal (99.9999%) and high purity demineralized water. The shape
and size of the nanoparticles were inspected by transmission electron microscopy (TEM)
using a JEOL JEM-1220 TE microscope at 80 KeV (JEOL Ltd., Tokyo, Japan), with a Morada
11 megapixel camera (Olympus Corporation, Tokyo, Japan) (Figure 1). The diameters of the Pt particles ranged from 2 to 19 nm. A sample of Pt for TEM
was prepared by placing droplets of the hydrocolloids onto Formvar-coated copper grids
(Agar Scientific Ltd., Stansted, UK). Immediately after drying the droplets in dry
air, the grids were inserted into the TE microscope (Figure 1). The zeta potential of the nanoparticle hydrocolloids was measured by electrophoretic
light-scattering method, using a Zetasizer Nano-ZS90 (Malvern, Worcestershire, UK).
Each sample was measured after 120 s of stabilization at 25°C in 20 replicates. The
mean zeta potential of the Pt nanoparticles was −9.6 mV.

Embryo model

Based on Polish law Article 2 of the act dated 21 January 2005 concerning the experiments
on animals (journal of law is dated 24 February 2005), there is no need to submit
an application to the local ethics committee for issuing an opinion about studies
where the chicken embryo is used. According to this act, chicken embryo is not definite
as the animal. Fertilized eggs (n = 150; 56 ± 2.2 g) from hens of the Ross line were obtained from a commercial hatchery
and stored at 12°C for 4 days. After 4 days, the eggs were weighed and randomly divided
into six groups (n = 25 eggs per group). The control group was not treated, while the other groups were
treated with 1, 5, 10, 15, or 20 μg/ml of NP-Pt solutions. The experimental solutions
were given in ovo by injection into the albumen (at two-thirds of the egg's height from the blunt end)
using a sterile 1-ml insulin syringe. Injection consisted of 0.3-ml NP-Pt hydrocolloid.
The injection holes were sterilized, and the eggs were then incubated at 37.5°C and
60% humidity and were turned once per hour for 19 days.

At day 20 of incubation, the embryos were sacrificed by decapitation. Embryos and
organs (brain, heart, liver, spleen, bursa of Fabricius) were weighed and evaluated
by Hamburger and Hamilton [18] (HH) standards.

Brain morphology: examination of brain tissue microstructure

Chicken brains (n = 12), three from the control group and nine from groups treated with 1, 10, and
20 μg/ml of NP-Pt solutions, were sampled and fixed in 10% buffered formalin (pH 7.2).
Fixed samples were dehydrated in a graded series of ethanols, embedded in Paraplast,
and cut into 5-μm sections using a microtome (Leica RM 2265, Leica, Nussloch, Germany).
The morphology of the chicken brains was examined using hematoxylin-eosin staining.
Proliferating cells were identified via immunohistochemistry using antibodies directed
against proliferating cell nuclear antigen (PCNA) [19]. Apoptotic cells were detected using rabbit polyclonal anti-caspase-3 antibody (C8487,
Sigma-Aldrich Corporation, St. Louis, MO, USA). Sections for this purpose were incubated
for 1 h with the rabbit polyclonal anti-caspase-3 antibody at room temperature and
were visualized with Dako EnVision+System-HRP (Dako K 4010, Dako A/S, Glostrup, Denmark), while further procedures were
identical as for PCNA detection. The proliferation and apoptosis levels were expressed
as the number of PCNA-positive cells and caspase-3-positive cells in the chicken brain
cortex, respectively (the area counted was 3,500 μm2).

Examination of brain tissue ultrastructure

Brain tissue morphology was examined by TEM. The tissues were fixed for TEM in fixative
consisting of 1% glutaraldehyde in PBS at pH 7.2. After fixation, the tissues were
post-fixed in 1% osmium tetroxide and dehydrated in a graded series of ethanols. The
tissues were embedded in a mixture of Araldite and Epon. Ultrathin sections (100 nm)
were cut on an ultramicrotome (EM UC6, Leica). The samples were viewed using a JEM-1220
TE microscope at 80 KeV (JEOL Ltd.), with a Morada 11 megapixel camera (Olympus Corporation).

Statistical analysis

Data analysis was carried out by monofactorial analysis of variance, and the differences
between groups were tested by multiple range Duncan test using Statistica version
10.0 (StatSoft, Tulsa, OK, USA). Differences with P < 0.05 were considered significant.

Results and discussion

Results

Growth and development

Embryo visualization did not show any genetic defects among the groups. Furthermore,
comparison with HH standards showed that all embryos had developed normally. Survival,
body weight, and weight of the brain, heart, spleen, and bursa of Fabricius were not
significantly different between all the groups (Table 1). However, the weight of the liver was significantly different in some NP-Pt groups
compared to the control group. None of the biochemical indices measured in the blood
sera of the embryos showed significant effects of the treatments (Table 2).

Table 1.Survival, body weight, and selected organ weight in control and groups treated with
different NP-Pt concentrations

Table 2.Activities of biochemical indices in the control and in groups treated with different
NP-Pt concentrations

Brain morphology: examination of brain tissue microstructure

Cell numbers in the brain cortex (area counted 3,500 μm2) were not significantly different between the groups (Table 3). However, histological evaluation of brain morphology revealed pathological changes
in the brain structure in embryos treated with NP-Pt, showing a moderate degradation
of the cerebellar molecular layer, neuronal loss in the cerebellum cortex, and astrocytosis
(Figure 2).

Table 3.Numbers of cells in the brain cortex in the control and in groups treated with different
NP-Pt concentrations

Examination of brain tissue ultrastructure

TEM examination of brain tissue morphology showed no abnormalities in the control
group. However, in embryos treated with NP-Pt, degradation of the mitochondria, rounded
nuclei with dispersed chromatin, and vacuoles in the cytoplasm were seen (Figure 3).

Immunohistochemical measurements showed that the number of PCNA-positive nuclei significantly
decreased after in ovo injection of NP-Pt solutions, attaining the lowest value in the 20-μg/ml group (Figure 4). Immunodetection of PCNA-positive nuclei by immunohistochemical methods was carried
out in cross sections of the granular layer of the cerebellar cortex. PCNA-positive
nuclei were brown, and PCNA-negative nuclei were blue (Figure 5). Immunohistochemical measurements showed the numbers of caspase-3-positive cells
significantly increased in the NP-Pt groups compared to those in the control group
(Figure 4). The greatest increase was observed in the group receiving 20 μg/ml of NP-Pt. Cross
sections of the granular layer of cerebral cortex were also immunostained with the
caspase-3 antibody. Caspase-3-positive cells showed brown cytoplasm, while the cytoplasm
of caspase-3-negative cells was blue (Figure 6).

Discussion

In the present work, we studied the effects of different concentrations of platinum
nanoparticle hydrocolloids administered to chicken embryos on their growth and development
as well as on the morphological and molecular status of the brain at the end of embryogenesis.
The chicken embryo is a very useful experimental model, developing without influence
of the maternal organism and allowing very fast and precise assessments of toxicity
[21,22]. Moreover, NP-Pt were administered at the beginning of embryogenesis, when, consequently,
nanoparticles could potentially penetrate the entire organism, including brain precursor
cells, differentiated cells, and brain structures, both before and after the appearance
of the BBB [7].

Our studies demonstrated that NP-Pt injected into eggs at concentrations of 1, 5,
10, 15, and 20 μg/ml did not influence the growth and development of the chicken embryos.
Their survival as well as examination of their morphology according to HH standards
of chicken embryo development did not differ between the control and NP-Pt groups.
No overt abnormalities that could indicate mutagenic effects of NP-Pt were observed.
These results are in agreement with a recent investigation demonstrating no toxic
effects of NP-Pt on the growth and development of Danio rerio embryo [13]. Furthermore, they are in agreement with our own previous studies regarding the effects
of nanoparticles of silver, silver/palladium alloy, and gold, showing no harmful effects
on growth and development of embryos when the nanoparticles were used at concentrations
below 100 μg/ml [23-27]. In contrast to NP-Pt, platinum-based drugs such as cis-dichlorodiammineplatinum (II) (cisplatin) do show toxic effects on the development
and mortality of rat embryos [28]. Platinum compounds also have toxic effects on mouse embryo development during organogenesis
and histogenesis [29].

In our experiment, body weight and the weights of selected organs in the chicken embryos
were not significantly affected by NP-Pt injection; however, liver weight was generally
lower in the NP-Pt groups compared to the control group, which might indicate some
harmful effects of NP-Pt. Subsequently, we measured the activities of hepatic enzymes
in blood serum (ALT, AST, and ALP) as markers of the functional and morphological
state of the liver [5], but these indices were not affected by NP-Pt. Consequently, our preliminary observations
regarding growth and development suggest that NP-Pt do not seem to be harmful when
evaluated at the whole body and organ level; however, potential subclinical changes
might occur at the tissue and molecular levels.

The chicken embryo is a suitable model to study neurotoxicity because the BBB is fully
developed and functioning after 15 days of incubation [7]. The key role of the BBB is protecting the brain from toxic substances. On the other
hand, the blocking role of the BBB is problematic because drugs used to treat many
diseases of the central nervous system are unable to cross this highly specific barrier
[30]. Application of NP-Pt at the beginning of embryogenesis makes it possible for NP-Pt
to penetrate different tissues, including brain precursor cells and structures. Moreover,
enclosed and separated from the mother and environment, the organism has no possibilities
to remove the nanoparticles from the egg, and consequently, the embryos were permanently
exposed to PN-Pt during 20 days of embryogenesis (before and after BBB occurrence).

The present results demonstrated that PN-Pt did not cause any difference in brain
weight evaluated at the end of embryogenesis. Histological assessment of the brain
structure revealed some minor pathological changes, but the number of brain cortex
cells was not affected. However, more detailed examination of the brain tissue ultrastructure
indicated some neurotoxic symptoms from NP-Pt treatment, including deformation and
degradation of the mitochondria. Similar results were obtained for cisplatin, showing
mitochondrial and nuclear DNA damage in the dorsal root ganglia [31]. Thus, not only platinum salts but also NP-Pt, via mitochondrial disruption, may
lead to mitochondria-dependent apoptosis. Although almost half the neuronal cells
die by apoptosis during normal brain development, this physiological process may be
enhanced under toxic conditions [32]. However, the stimulation of mechanism of apoptosis within tumor cells is considered
a highly advanced cancer therapy [33] and, in this respect, NP-Pt can enhance the apoptosis of cancer cells.

Cytochrome c released from the mitochondria into the cytosol is one of the first steps
in the mitochondrial apoptotic pathway. Cytochrome c and ATP are bound to the apoptotic
protease-activating factor-1 [34]. The merger of these two structures creates an apoptosome and activates caspase-9.
Active caspase-9 is responsible for the activation of the executioners, caspase-3
and caspase-7 [32,35]. We examined the activity of caspase-3 to detect apoptosis. Our results showed an
increasing level of caspase-3-positive cells in chicken brain samples from groups
treated with NP-Pt. These results agree with studies suggesting that platinum-based
drugs trigger DNA damage, which induces apoptosis with the activation of caspase-3
[36,37].

Opposing apoptosis is the process of cell proliferation, and thus, the interaction
between apoptosis and proliferation, observed after platinum-based drug treatment,
is a key factor in cancer therapy [38]. To examine the status of proliferation after NP-Pt treatment, we also identified
the level of PCNA-positive nuclei in the brain tissue. The immunohistochemical analyses
showed a decline in PCNA-positive nuclei in the NP-Pt groups. PCNA is a key factor
in the replication of genetic material and is involved in the cell cycle and proliferation
processes [39]. This may indicate that NP-Pt analogs to platinum-based drugs, where Pt exists in
cationic form, activate apoptosis and at the same time suppress proliferation. However,
the toxic side effects of NP-Pt seem to be much smaller than those caused by platinum-based
drugs containing ionic Pt. This may suggest that NP-Pt could be used in cancer therapy
instead of ionic Pt, especially for brain cancer, because the particles can pass the
BBB and reach the tumor tissue in the brain.

Conclusions

Platinum nanoparticles administered to chicken embryos at the beginning of embryogenesis
at concentrations of 1 to 20 μg/ml did not affect the growth and development of the
embryos. Examination of neurotoxicity after NP-Pt treatment showed no changes in the
number of cells in the brain cortex; however, analyses of brain tissue ultrastructure
revealed mitochondria degradation. NP-Pt activated apoptosis as well as decreased
the rate of proliferation of the brain cells. These preliminary results indicate that
properties of NP-Pt might be utilized for brain cancer therapy, but potential toxic
side effects must be elucidated in extensive follow-up research.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

MP carried out in ovo studies and drafted the manuscript. ES conceived the study and helped draft the manuscript.
SJ participated in the analysis of biochemical indices. TO participated in the histological
studies and helped draft the manuscript. MK participated in the immunohistological
studies. MG participated in the design the experiment. MW participated in the statistical
analysis. AC participated in the design and coordination and helped draft the manuscript.
All authors read and approved the final manuscript.

Authors’ information

MP is a PhD student at the Warsaw University of Life Sciences (WULS). ES has PhD and
DSc degrees and is a professor and head of a department at WULS. SJ is a PhD student
at WULS. MG has PhD and postdoctorate degrees at WULS. TO has PhD and DSc degrees
and is a professor and head of a department at WULS. MK has PhD and postdoctorate
degrees at WULS. MW is a PhD student, and AC has a DSc degree and is a professor and
head of a division at the University of Copenhagen (UC).

Acknowledgments

This work was supported by grant NCN 2011/03/B/NZ9/03387. This report is part of Marta
Prasek's PhD thesis.